The emergence of drug-resistant tumor cells is a major limitation to the successful treatment of cancer. Trans-Lesion Synthesis (TLS) is a major mechanism by which cancer cells acquire DNA damage tolerance and resistance to front line genotoxic chemotherapies such as cisplatin. Cisplatin induces bulky intra-strand DNA adducts that cause DNA replication fork stalling and lead to cell death. TLS involves the recruitment of specialized TLS DNA polymerases to stalled replication forks. Due to flexible active sites, TLS DNA polymerases (such as DNA polymerase eta or Pol?) can replicate damaged genomes and maintains replication fork progression, thereby conferring DNA damage tolerance. Because Pol?-mediated TLS is a major mechanism by which cancer cells evade chemotherapy, the long-term goal is to develop small molecule inhibitors of TLS to improve clinical approaches to cancer treatment. The Specific Aims of this project are: (1) To validate novel small molecule inhibitors of TLS for cancer chemotherapy [(2) To define a novel mechanism of DNA damage tolerance and chemoresistance in cancer cells.] In support of SA1 we have developed a novel and sensitive High-Throughput Screening (HTS) assay for measuring association between Pol? and ubiquitinated PCNA (an interaction that is essential for TLS and cisplatin tolerance in cancer cells). We have performed HTS of compound libraries and have identified candidate inhibitors of the Pol?-PCNA interaction. In the proposed work we will validate those candidate small molecules as inhibitors of the Pol?- PCNA interaction and evaluate the utility of these inhibitors as chemosensitizers in cultured cancer cells. [ In support of SA2 we have identified a Cancer/Testes Antigen termed MAGEA4 as a proximal activating component of the TLS pathway in lung cancer cells. Therefore, experiments in SA2 seek to define the exciting, new mechanism by which MAGEA4 promotes TLS and confers DNA damage tolerance in cancer. We will test the hypothesis that MAGEA4 stimulates TLS via an interaction with the E3 ubiquitin ligase, Rad18, an upstream regulator of Pol? activity. The hypothesis that MAGEA4 mediates DNA damage tolerance and chemoresistance is a paradigm-shifting discovery and if proven will advance the therapeutic approaches in MAGEA4-expressing, chemoresistant tumor cells. Owing to the cancer cell-specific expression of MAGEA4, the MAGEA4-Rad18- Pol? signaling axis represents an attractive, druggable target. The proposed work is significant because it seeks to improve traditional cancer therapies through the innovative application of personalized medicine for DNA damage-resistant cancer cells.